Production of plasma



Patented May 5, 1942 PRODUCTION OF PLASMA Ivan A. Parlentjev, Nyack, N. Y., assignor to Lederle Laboratories, Inc., New York, N. Y a

corporation of Delaware No Drawing.

Serial No. 300,595

Claims.

This invention relates to improvements in the methods of bleeding immune animals and is more particularly directed to the production of increased yields of plasma.

In the production of antitoxins and the like, the customary procedure entails the bleeding of immunized animals such as horses, pigs, rabbits, etc. For bleeding as serum, the blood is allowed to flow into a suitable vessel where it is permitted to coagulate and clot, subsequently separating a liquid fraction which is removed from the clotted material as serum. The yield of serum is usually 40% to 50% by volume of the original blood.

To obtain plasma, the blood is led into a receptacle containing a suitable anticoagulant which maintains the fibrin in solution. The blood does not coagulate but the blood cells settle to the bottom of the vessel and transparent plasma collects in the layer over the blood cells. The plasma layer may be siphoned off and treated as desired. As anticoagulants, one customarily uses potassium oxalate, sodium citrate and the like. In an efficient processing the yield of plasma represents about 60% to 70% of the volume of the original blood, and in some cases, in the bleeding of hogs, plasma bean extract has been used to pack the blood cells more firmly and the yield of plasma may increase to as much as 80%.

It is one of the objects of my invention to facilitate the treatment of the blood in order to increase the yields of plasma.

It is a still further object to facilitate the pro duction of plasma without destroying any of the immune property thereof.

These and other objects are primarily obtained by the use of sodium pyrophosphate as an anticoagulant for the production of plasma. Bleeding into a vessel containing sodium Pyrophosphate as an anticoagulant and washing the Application October 21, 1939,

blood cells increases the yield of plasma from 20% to 30% over the yield obtained with the usual bleeding as oxalated or citrated plasma, or an increase of 40% to in yield over the usual methods for bleeding as serum. The product contains the same degree of immune property (number of protecting units per gram of nitrogen) as the prior methods.

In bleeding to obtain plasma, I generally prepare an aqueous solution containing about 1% 01 sodium pyrophosphate and about 1.5% of sodium chloride. This solution is adjusted to a pH of 7.0 to 7.2 with phosphoric acid, monoor disodium phosphate. To a suitable quantity of this solution, an equal volume of blood is added. This mixture is kept in the cold room for twenty-iour hours after which the supernatant plasma is siphoned oil from the settled blood cells. To obtain additional plasma, the residue of blood cells is centrifuged or passed through a cream separator. centrifuging gives a good product without any hemolysis. The use of a cream separator is somewhat faster than the centrifuge method but with the former, the liquid obtained is sometimes dark colored, indicating hemolysis, breaking down of some of the blood cells. The

blood cells may also be washed by a suitable washing method such as will be described.

In order to avoid dilution, I may utilize much less of the anticoagulant solution so that only 1 6 as much solution is used as compared with the added blood. More specifically, I may add 8 liters of blood to 800 cc. of a 5% sodium pyrophosphate solution containing 1.5% sodium chloride, the pH of the solution having been adjusted to 8.2 prior to the addition of the blood. Alter twenty-four hours in the cold room, plasma is siphoned on and to the remaining blood cells, there may be added an equal volume of washing solution containing 0.5% sodium pyrophosphate and 1.5% sodium chloride. the solution having been adjusted to a pH 7 prior to use. The washing liquid may be separated from the blood cells by centrifuging or by use of a cream separator COMPARISON or BLEEDING Houses in Praornospnn'rn Diphtheria horses Oxamra AND as described above.

fhe washing solution is greatly improved if 111mm Horse Volume Uni or laid by there 18 added to the solution a small amount oi nnm- Dilucnt or blood a beedingln a gum. A typical washing solution contains 0.5% take p osphate SOdlllm pyrophosphate, 0.85% sodium chloride and 0.5% gum tragacanth. The solution is ad- Lam justed to pH 7-7.5 prior to use. The presence of 7,855 Pot. oxalate 8 3,022,500 35 the gum facilitates sedimentation or the blood 5 33 5335 53 3??? g 333% cells and assists in separation of the liquid go yro l ios hoee g :,g 0 33 o .oxa a e plasma from the cells 7,901 Na pyrophosphate 16 8,612,000 29 In place of the gum tragacanth, I may use 7,903 Pot. oxalate 4 1,020,000 22 0.5% to 1.0% gum acacia. gum arable and the igggggg fi f g 'gtgffi like I have found that 0.2% or even less of 7,863 Napyrophosphate 10 7,488,000 tragacanth is effective. The use of gum tragecanth broadly in the treatment of blood for obflu gnaryg l taming plasma is claimed in my copending applig gg gggg i g 2 cation Serial No. 300,594. filed October 21, 1939. 90

Staphylococcus horses .Volume U t infield H rs number Diluent of blood an by b ceding taken I Liter: Plrce'nt 7234,7001,700s.774s Potassium oxalate 10 1,009,500 7234, 7601, 7608, 7748 Sodium pyrophosphate- 16 l 265 970 5.15, 6737, 7025, 7233. 7604. Potassium oxalate. 32 2,208,000

7007, 7724, 7750. 29 5715, 6737, 7025, 7233, 7004, Sodium pyrophosphate 32 2, 864, 250

Summary:

Potassnm' oxalate 3, 217, 500 28 Sodium pyrophosphate 4, 130, 220

The use of sodium pyrophosphate as the anticoagulant for the blood has the further advantage that it is possible to wash the blood cells after the supernatant plasma has been separated from the settled cells. When oxalate or citrate is used as an anticoagulant, the blood cells are very fragile and any washing treatment causes a breaking up of the cells with bad hemolysis. When sodium pyrophosphate is used, the blood cells, after the siphoning of the plasma, can be washed with isotonic or hypertonic salt solutions without noticeable hemolysis. The use or the pyrophosphate also serves to prevent oxidation and especially oxidation due to the presence of iron in the blood.

The following is typical of the results obtained with sodium pyrophosphate and indicates clearly the increase in yield obtained as compared with potassium oxalate as the anticoagulant.

BLEEDING m DRY AND LIQUID ANTIC'OAGULANT The increase of yield of antitoxin by bleeding in sodium pyrophosphate was achieved by bleed- 0 ing in equal volume of diluent (sodium pyrophosphate), or in cases in which solution of sodium pyrophosphate was used in the amount of 11% against the volume of drained blood, the blood cells after siphoning of plasma were washed with salt solution (0.85%) containing 0.5% sodium pyrophosphate. The washing liquid was used in an amount equal to the volume of settled blood cells. Cells suspension was passed through cream separator, or cells settled themselves by the addition to washing liquid of 0.5% gum of tragacanth The following indicates the eilectiveness of my method utilizing a dry anticoagulant wherein the dry sodiumpyrophosphate or the like is placed in a suitable vessel and the blood is added. In calculating the drop of plasma potency as compared with serum potency, allowance for dilution of plasma by anticoagulant was made.

v 1 Percent drop Material Antioo mfll t ig g Potency 12 5:253 Note to serum S 0:. 5O Un its Pei-ran 875 00 T e i: an n s Plasma My 120 1118' N 8 pyr phosphate, 25 mg. 60 900 u D2 1 I horse N 0. pm. Na phosphate. 7622. do 2.7% sol. 120 mg. Na pyrophosphate, 25 45 725 D0 mg. prim. Na phosphate, 5 cc. 1% N 001. do Dry 50 mg. 1% potassium oxalate 50 900 U Tm do..- 171,;I so(l).l50 mg. potassium oxalate, 5 cc. 1% 45 7 g In a i do Dry 100 mg. Na citrate 5 350 3 7 do Sol. 100 mg. N a citrate, 6 cc. 1% N 001-... 725 3 1 serum" Fiocculg.

' tion N-sl, Di htheria Plasma. Dry :mg. Na pyrophosphate 10 mg. 50 I l 04' N01 .ra s am. 4 l 0204. o o mg. a a 0, mg. 5 87 2 prim. Na phosp ate, 5 cc. 1% N 1101. 1 i

BLEEDING m DRY arm LIQUID Amcoaomm-Con.

. Percent drop Material Anticoagulant gm Potency Note to serum Cc. Unite Percent D i phtherls, Plasma Dry 200 mg. prim. Na phosphate, 700 mg. 50 970 12 Fioeculahorse No. second Na phosphate. tion test. 8284. ...do.. Sol. 200 mg. prim. Na phosphate, 700 mg. 45 870 13 Do.

second Na phosphate, 5 cc. 1% NaCl. do Dry 50 mg. potassium oxalate 50 1,050 5 Do. do..-.- S 1 58 mg. potassium oxalate, 5 cc. 1% 45 B85 11 Do.

a do.. Sol. 50 mg. germanln, 5 cc. 1%NaCl-. 45 88] 11 Do.

The above-described samples were tested from horse #8284 for potency on guinea pigs, with the following results:

Estimated Material Anticoagulant Potency mm Bernm.- 1,000+ 1,200 3.5 1,100 Plasma" Dry Na pyrophos- 1,000+ 1, 100 3.5 1, 050

p a Do..-.. 801.11g pyrophos- 900+ 5.5 950 p e. Do..... Dry mixturegmm. 900+ 1,000 3.5 950 and secon Na phosphate. Do.-. Drly potassium ox- 1,100+ 5. 5 1,100

a a e. Do Sol. potassium ox- 900+ 1, 000 3. 5 950 alate.

' Potency Material Anticoagulant test floecuin blood i pig in Er. pig test Cc. Percent Percent Serum 50 830 900 Diphtheria, Plasma Dry mixture 120 Na pyrophosphate, 100 mg. prim. 50 775 850 7 6 horse N 0. Na phos. 8284. do Sol. 5 cc. Na pyrophosphate, 1% NaOl H7. 2 45 025 700 17 14 do.. Sol. 5 cc. 1% potassium oxalate and 1 0 NaCl 45 655 725 13 11 V. P 's o ume 0- one Material Ant.coagulant of blood tency Y plasma Cc. Percent Serum. 50 59 Diphtheria, P1asma.- Dry mixture 120 mg. Na pyrophosphate, 100 mg. prim. Na phos 50 545 9 horse No. do..- Sol. 5 cc. 5% Na pyrophosphate, 1% NaCl prim., pH 7.2 45 440 18 3284 ...do.., Dry mixture 200 mg. prim. Na phos. and 700 mg. second Na phos. 50 495 17 Serum. 50 60 Diphtheria, Plums... Dry mixture 120 mg. Na pyrophosphate, rng. prim. Na phos 50 55 0 gisorse No. do. 5011 7112? Na pyrophosphate, 25 mg. prim. Na phos., 5 so. 45 35 do. Dry potassium oxalate 50 mg 50 55 9 do Sol. 50 mg. potassium oxalate, 5 cc. 1% NaC 35 35 do Dry 100 mg. Na citrate 9 do Sol. mg. Na citrate, 5 cc. 1% Nat] 45 35 35 Serum. ii 50 000 Diphtheria, Plasma" Dry mixture 720 mg. Na pyrophosphate, 100 mg. prim. Na 50 830 3 horse No. phosphate. 8200. --.do..... Sol. mg. Na pyrophosphate, 100 mg. prim. Na phos., 5 cc. 45 755 7 1% NaCl.

Serum- 50 445 0 Dgphthelrzia, Plasma Dryl'lntiixture 120 mg. Na pyrophosphate, 25 mg. prim. Na pins 50 445 0 orse o. a e.

. .do. .809. 120 mg. Na pyrophosphate, 25 mg. prim. Na phos., 5 cc. 45 380 6 1% NaCl.

Percent v 1 3 i N t c s o ume ecrease o o e Material Anticoagulant of blood tency potency in test type H plasma toward serum 11 Ce. Units Percent 50 250 P n e u m o rymixture 120 mg. Na pyrophcsphate, 25 mg. 50 300 20 Increase horse N o. prim. Na phos.

" Sol. 120 mg. Na pyrlolphosphate, 25 mg. prim. 45 275 22 Do.

Na phos. 5 cc. 1% e01. Dry second Na phos. 1050 mg 50 275 10 Do. Dry potassium oxalate 50 mg 50 325 30 Do. Sol. 50 m potassium oxalate, 5 cc. 1% NaCl--. 45 300 33 Do. Dry Na c trate 100 mg 50 300 20 Do, Sol. 100 mg. Na citrate 5 cc. 1% NB-C1...-..-.. 45 275 22 Do.

TYPE II PNEUMO Hossn One 0. S. test showed 60.

justed to pH 8.3 to 8.8 prior to the addition of the blood since the bufler action or the blood readily compensates for the alkalinity of the pyrophosphate solution since the p r phosphate is alkaline. The pH adjustment may be made by the addition 01' concentrated phosphoric acid, monobasic (primary) sodium phosphate or dibasic (secondary) sodium phosphate, themonosodium phosphate usually being preferred when the pyrophosphate is used in the dry form. In this case the pyrophosphate is best used as powder and the monobasic sodium phosphate in very small crystals. Positive anticoagulating effect on blood is obtained by final concentration of phosphates as low as 0.3%. The solutions which BLEEDING Srsrmzrooooous Hoasns m DRY AND WET Sonnm Prnornosrm'm e "a Horse Volume Volume Pocasgs bled 'i Anticoagulant of blood of plasma tency in solution oi Na pyrophosphate 5, 715 400 cc. sol. 1 0 NaCl 8% Na pyrophosphate prim. 8.8 4,000 2, 600 5, 715 Dry mixturZlO gm. Na pyrophosphate, 2 gm. prim. N a phosphate 4, 000 2, 400 7, 122 400 cc. s01. Na pyrophosphate (see above).-- 4, 000 2, 900 7,122 Dry mixture Na pyrophosphate (see above) 4, 000 2, 700 7, 224 400 cc. sol. Na pyrophosphate 4, 000 2, 600 7, 224 Dry mixture N a pyrophosphate 000 2, 500 7, 604 400 cc. sol. Na pyrophosphate.. 4, 000 2, 600 7, 604 Dry mixture Na pyrophosphate. 4, 000 2, 600 7, 608 400 cc. sol. Na pyrophosphate.- 4, 000 2, 700 7, 608 Dry mixture N a pyrophosphate 4, 000 2, 800

Horse #56K was bled out in dry sodium pyrophosphate. Potency of serum, 90 units; potency of plasma, 100 units.

The preceding tables contain comparisons of potencies for sera and plasmas prepared with different anticoagulants. For close comparison, sera and plasmas in each experiment were taken from small portions of the same bleeding. As can be seen from the tables, the potency of the material is, in many cases, a little higher in plasma prepared from dry anticoagulant. Using anticoagulant solution, plasmas were obtained that, after allowing for the dilution, were of the same potency as serum or, in some cases, slightly lower. Even in such cases, when using the solution of sodium pyrophosphate, the total yield of potency represents a considerable increase, due to a more complete recovery of plasma achieved by the washing process. The use of sodium pyrophosphate as an anticoagulant increases the yield of plasma content from to over the customary bleeding using oxalates or cifirates, and an increase of to 50% over the bleeding as serum.

The pyrophosphate has the further advantage that the blood cells are in such condition as to permit washing thereof without substantial hemolysis. The pyrophosphate also servesto prevent oxidation of not only the blood, serum and refined antitoxin. The pyrophosphate is sufiiciently eifective as an anticoagulant so that only 10% of the pyrophosphate solution may be used as compared with the volume of drained blood. Following the washing of blood cells with an equal volume of washing liquid, the total volume of pooled liquid (plasma washings) is equal to or only slightly larger than the original volume of drained bood.

It wil be further noted that the use of dry sodium pyrophosphate produces higher potencies of the order of 5% to 10% greater than when the blood is drawn into solutions of anticoagulants.

The sodium pyrophosphate il preferably adplasma but also whole prises mixing blood with dry are used are previously sterilized as in an autoclave at 20 pounds pressure for thirty minutes. The dry mixtures may be sterilized by dry heat at -200 C.

It will be apparent that my invention is subject to some change or modification in the details set forth above without departing from the spirit and scope thereof.

I claim:

1. The process of producing plasma which comprises mixing blood' with sodium pyrophosphate as an anticoagulant, allowing the mixture to stand until the blood cells settle out, separating the supernatant plasma from the blood cells and subjecting the blood cells to centrifugal action to remove additional plasma associated with the blood cells.

2. The process of producing plasma which comsodium pyrophosphate as an anticoagulant and separating plasma from the blood cells.

3. The process of producing plasma which comprises mixing blood, dry sodium pyrophosphate and dry sodium phosphate and separating plasma from the blood cells.

4. The process of producing plasma which comprises mixing blood with an aqueous solution at about pH 8 and containing sodium pyrophosphate and sodium chloride, allowing the mixture to stand until the blood cells settle and separating the supernatant plasma from the blood cells.

5. The process of producing plasma which comprises mixing blood with sodium pyrophosphate as an anticoagulant, allowing the mixture to stand until the blood cells settle out, separating th supernatant plasma from the blood cells and washing the blood cells with a solution containing sodium pyrophosphate and gum traga canth.

IVAN A. PARFENTJEV. 

